Iron is a vital element required by all microorganisms except Lactobacilli and Streptococcus sanguis. The ability to acquire iron in several pathogens has been related to their virulence. Some iron-transport systems also naturally transport antibiotics. Thus iron-transport systems could either be blocked or exploited to deliver antibiotics to control pathogens' growth inside the body of the host. Under iron-depleted conditions many gram-negative bacteria including several pathogens, express complex iron transport systems. These systems consist of: 1. An iron-chelating molecule called a siderophore, 2. A specific outer membrane receptor protein recognizing iron-siderophore complexes, 3. A periplasmic binding protein, 4. An ATP dependent ABC type transporter and 5. TonB-ExbB-ExbD complexes located in the inner membrane. The bound iron-siderophore complexes are transported through the outer membrane receptors in an energy dependent manner. The energy is presumably transduced by the TonB complex, which utilizes the cytoplasmic membrane potential. The binding of the iron-siderophore complex has been well characterized but the mechanism of the transport of the bound siderophore across the receptor is poorly understood. The long-term goal of this project is to understand the mechanism of iron transport at the molecular level. This knowledge could help to control bacterial infections by disrupting iron transport and also to design novel antibiotics to be delivered through these systems. A combination of genetic, biochemical and crystallographic methods will be used. The aim of this project is to identify the amino acid residues specifically involved in the transport process and not in binding using the receptors FepA and FhuA in Escherichia coli. This will be done by: 1. Identification of the conserved regions through multiple sequence alignment of the 19-receptor proteins transporting ferric siderophores. These residues will be mutated and analyzed first in FepA to identify mutants with normal binding and defective transport; 2. The corresponding residues showing normal binding and defective transport will be mutated in FhuA; 3. The residues believed to be involved in TonB interaction will be tested for their ability to physically interact with TonB by formaldehyde cross-linking and also for their TonB dependence using a TonB strain; 4. For the mutant proteins showing normal binding and defective transport, methods of purification and crystallization will be standardized.